Solid-state nanopores have been developed in order to sense molecular analytes and in particular biomolecules such as DNA, RNA and proteins because they are viewed as being more robust and less dynamic than biopores which are based on protein (toxin)-lipid membrane systems (Clarcke et al., 2009, Nat. Nanotechnol., 4, 265). Solid-state nanopores can operate in various liquid media and pH conditions and their production is scalable and compatible with nanofabrication techniques and can be integrated with other sensing methods that exploit tunneling or local potential gating.
The sensing principle is the same as in bio-engineered pores and ideally, the sequence of nucleotides, genetic information, along a single DNA molecule can be registered by monitoring small changes in the ionic current caused by the transient residing of single nucleotides within a nanometer size pore.
Before sequencing with solid state nanopores, three fundamental requirements on ionic or transverse current signals have to be met: spatial resolution in the order of few nucleotides, high signal to noise ratio to distinguish between signals related to different nucleotides and temporal resolution of the signal that allows acquiring enough points per nucleotide using state of the art current-voltage amplifier systems have to be achieved.
However, solid state nanopores exhibit a relatively lower single molecule detection sensitivity compared to biopores due to their intrinsic thickness and lack of control over surface charge distribution: temporal resolution of ionic current in solid state nanopores is on order of 10-50 base pairs/ms (Branton et al., 2008, Nat. Nanotechnol., 26, 1146). Together with lower ionic current signal to noise ratio, relatively larger sensing region, which is due to the pore membrane thickness, has been major obstacle in achieving sequencing data when using solid state nanopores.
Recently, thin membranes have been proposed to extend the applications of solid-state nanopore to, e.g., detection of short DNA oligomers and differentiation of short nucleotides homopolymers (Venta et al., 2013, ACS Nano, 7, 4629-4636). Several groups have used monolayer graphene (thickness of ˜0.35 nm) as a nanopore membrane for the detection of DNA translocation (Garaj et al., 2010, Nature, 467, 7312, 190-193) and simultaneous detection of DNA translocation with two synchronized signals, the ionic current in the nanopore and local potential change in the graphene nanoribbon transistor has been reported (Traversi et al., 2013, Nat. Nanotechnol., 8, 939-945).
However, graphene nanopores exhibit strong hydrophobic interactions with DNA that limits their long-term use due to the clogging. Schneider et al. have implemented surface functionalization with pyrene ethylene glycol of graphene nanopores and demonstrated that this process prevents DNA absorption on graphene and renders graphene nanopores usable for extended periods of time (Schneider et al., 2013, Nat. Commun., 4, 2619).
Several attempts have been carried-out for improving temporal resolution of translocating DNA molecules in solid-state nanopores, including using the DNA pore interactions, controlling the electrolyte parameters (temperature, salt concentration, viscosity), nanopore surface functionalization, change in nanopore surface charge though light-control and applying an electrical bias voltage across the nanopore to reduce the mobility of the DNA. Fologea et al. (Fologea et al., 2005, Nano Lett, 5, 1734) added glycerol into buffer to increase the viscosity and consequently reduce the mobility of DNA and by controlling the electrolyte temperature, salt concentration, viscosity, electrical bias voltage across the nanopore, obtained a 3 base/ms, but working with glycerol/water reduced the ionic current signal. In addition, the highest viscosity of the solution that they could use was 5.2 cP.
In conclusion, DNA translocations in biological nanopores are currently too slow, on the other hand in solid-state nanopores are too fast compared to the optimal DNA sequencing velocity of 1-50 nucleotide/ms (Venkatesan et al., 2011, Nature Nanotechnology 6: 615-624). So far achieved temporal resolution in solid-state nanopores is on order of 3000-50000 nt/ms (Branton et al., 2008, supra).
Therefore, there is a need for selective and sensitive sensing systems for analytes, in particular biomolecular analytes that allow a rapid analysis at the molecular levels such as for DNA, RNA and protein sequencing.